The goal of this research is to understand chromatin mechanisms that maintain gene expression states during development. Molecular, genetic and biochemical methods will be used to study the Polycomb group (PcG) transcriptional repressors of Drosophila, which provide one of the premier models for deciphering chromatin mechanisms in development. PcG proteins control selective expression of homeotic (Hox) genes along the anterior-posterior (A-P) axis which, in turn, program differentiation of body structures. For normal body patterning to occur, Hox genes must be kept silent in A-P positions where they are not normally expressed. Hox expression patterns are set by segmentation gene products in 2-hour-old fly embryos, but these initial regulators decay by about 4 hours. The Polycomb group proteins then assume control to maintain Hox gene silencing during the rest of development. Thus, PcG proteins provide molecular memory of spatial cues present in early embryos. Current models suggest that PcG silencing is maintained through covalent histone modification and stable association of PcG protein complexes in the local chromatin. The PcG proteins, and the chromatin complexes they form, are highly conserved from flies to humans. Human PcG proteins play critical roles to maintain pluripotency of embryonic stem cells. Overabundance of PcG proteins is also implicated in disease progression in cancers of the breast, prostate, and other tissues. Their expanding importance in stem cell biology and cancer epigenetics underscores the need to understand basic PcG chromatin mechanisms. This project will determine PcG mechanisms using Drosophila, which provides one of the best-characterized systems for in vivo investigation of PcG silencing. This research will investigate molecular roles of PcG complexes and their subunits. Much of the work focuses on a PcG complex called PRC2 (Polycomb repressive complex 2). PRC2 has four core subunits and an enzyme activity that methylates histone H3 on lysine-27. One Aim is to determine how the noncatalytic subunits make key inputs to PRC2 function in vitro and in vivo. A second Aim addresses in vivo consequences of histone methylation and whether PRC2 has any function besides enzyme activity. A third Aim is to define the molecular role of another critical PcG repressor, called Sex comb on midleg (SCM), which appears to work independently of PRC2. The methods will include loss-of-function and over-expression studies, site-directed mutagenesis, transgene manipulation, chromatin immuneprecipitation, enzyme assays, protein purification and chromosome immunostaining. Fulfillment of these Aims should advance knowledge of basic PcG mechanisms and also of epigenetic processes that control human stem cell fates and that underlie certain human cancers. PUBLIC HEALTH RELEVANCE: This research is to determine how a set of highly conserved regulatory proteins, called Polycomb group (PcG) proteins, keep genes turned off during animal development. In humans, PcG proteins are critical for embryonic stem cell maintenance and they are implicated in breast cancer, prostate cancer, and cancers of other tissues. This research will advance basic understanding of gene regulatory mechanisms and provide knowledge that could impact stem cell applications in medicine and development of anti-cancer strategies.